Progressive multifocal leukoencephalopathy (PML), caused by human polyomavirus JC (JCV), remains an important cause of morbidity and mortality among HIV/AIDS patients even in the post-HAART era. The underlying mechanism(s) of JCV entry into the central nervous system (CNS) is poorly understood. That is, it is unclear if cell-free JCV uses sialic acid receptor (SAR) to infect human brain microvascular endothelial (HBMVE) cells or if JCV-infected B cells alter the expression patterns of cell adhesion molecules (CAM), such as very late antigen-4 (VLA-4) and lymphocyte function associated-antigen-1 (LFA-1), and chemokine receptors, CCR2 and CXCR1/CXCR2, to preferentially cross the BBB. Our preliminary and published data demonstrate that JCV productively infects primary HBMVE cells;cell-free JCV crosses the in-vitro BBB model;infectious JCV virions can survive latently in Epstein Barr virus-transformed B cells;and latent JCV from B cells can infect primary human fetal glial (PHFG) cells. However, data on the transmigration of JCV-infected B cells across the BBB are unavailable. Based on these data, we will test the following hypotheses: 1) cell-free JCV employs the SAR to infect HBMVE cells, and to cross the in-vitro BBB;2) latently infected B cells preferentially cross the BBB by modulating of the expression of CAM and chemokine receptors. The proposed research will specifically address gaps in our knowledge about the mechanism of transmigration of cell-free and B cell- associated JCV across the BBB by addressing two specific aims: Specific Aim 1: To characterize the interaction of cell-free JCV with the HBMVE cells and the in-vitro BBB, by documenting changes in infection and replication of JCV in HBMVE cells, and JCV transmigration across the BBB when treated with SAR blockers, and changes in BBB by monitoring the expression of junctional proteins, claudin-1 and -5 and occludin, TEER, and paracellular markers, such as inulin or dextran, before, during and after transmigration of JCV. Specific Aim 2: To demonstrate and characterize the trafficking of JCV-infected B cells across the in-vitro BBB by infecting B cells with JCV and comparing the expression of adhesion molecules VLA-4 and LFA-1 and chemokine receptors CCR2 and CXCR1/CXCR2 on JCV-infected and -uninfected B cells, and comparing the transmigration of JCV-infected and -uninfected B cells across the in-vitro BBB, and documenting changes in the in-vitro BBB before, during and after transmigration. CNS infections are difficult to treat because of the various barriers surrounding the brain. The proposed study should provide novel insights into how tight junction proteins and CAM become dysregulated and further facilitate migration of JCV across the BBB, and how specific inhibitors can block JCV from entering and/or infecting HBMVE cells, astrocytes and oligodendrocytes. Newfound knowledge may lead to improved therapeutic interventions for PML. PUBLIC HEALTH RELEVANCE Progressive multifocal leukoencephalopathy (PML), a fatal brain disease, caused by human polyomavirus JC (JCV) remains one of the important causes of mortality and morbidity among people with defective immune systems. Currently, there are no preventive or therapeutic options available to manage PML patients. The proposed research will utilize in-vitro systems to delineate cellular and molecular steps involved in JCV transmigration across the blood-brain-barrier and the discovery of such mechanisms may assist us in developing preventive or therapeutic intervention for this incurable disease.